Endothelial Cells as Biosensors for Occupational Cardiovascular Risk Engineered nanomaterials (ENMs) have an unknown toxic potential and the relationship between the biological effects and the physicochemical properties remains uncertain. Recent ENM inhalation studies have identified cardiovascular outcomes that may be relevant to human health. Specifically, inhalational exposure to nanoparticulates may drive the chronic development and acute exacerbation of clinically-relevant coronary arterial disease. Because of the myriad physicochemical permutations of ENMs, a systematic means of predicting / testing cardiovascular safety is needed. The present study is structured to develop a novel, translational in vitro model to assess systemic inflammatory changes caused by inhaled ENMs. Rather than biased approaches that measure discrete plasma components, we propose a novel paradigm whereby primary human endothelial cells (replete with receptors / responses to biochemical stimuli) are used to assess the complete inflammatory potential of the serum. This method of biosensing is feasible due to a commonality, primarily through NFB pathways, of endothelial cell response to numerous molecules. We have already used this assay paradigm to identify cardiovascular effects of inhaled diesel emissions and nitrogen dioxide in humans exposed under controlled conditions. Importantly, endothelial responses were far more robust than measures of inflammatory plasma cytokine usual suspects, such as CRP or TNF. The major goals of the present application are to develop this model for rodent species and explore the relative impact of inhaled multiwalled carbon nanotubes (MWCNTs) and graphene. Thus, in Aim 1, we will rigorously optimize an in vitro testing platform using three primary murine endothelial cell lines, focusing on expression of adhesion molecules and generation of NO. This will involve selection of a maximally-responsive cell line (derived from aorta, coronaries, or cerebral vessels) and characterization of responses to known inflammatory stimuli. We will also treat cells with serum from a known model of systemic inflammation to assess the range of responses. In Aim 2, we will assess serum-induced endothelial cell responses to serum obtained following pulmonary exposures of graphene and MWCNT. Exposures are ongoing with our collaborator at NIOSH (Erdely) and serum is being collected with this purpose in mind. All responses in endothelial cells will be related back to pathology and gene responses from the NIOSH work. The potential benefits of this biosensing assay include not only the ability to systematically compare toxicant potency in rodent models, but also as a translational diagnostic for early detection of workplace-associated injury or as a marker of therapeutic benefit for interventional strategies. We envision 3 areas where using endothelial cells as a biosensor of plasma inflammatory potential may have value: 1) diagnosis/prognosis of cardiovascular disease; 2) efficacy of therapeutic intervention; and 3) toxicology/safety testing of environmental contaminants/novel pharmaceuticals.